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Keywords:Mesenchymal stem cell M1 macrophage M2 macrophage Th1 Th2 Th17 Treg Immunomodulation A B S T R A C T Mesenchymal stem cells (MSCs) are strong immunomodulatory cells investigated in numerous clinical studies on fatal pathologies, such as graft versus host disease and autoimmune diseases; e.g., systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. Macrophages are one of the critical cells linking the innate and adaptive immune system, and it has been shown that MSCs can differentiate between pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype of macrophages. However, it has not yet been fully clarified whether these differentiated macrophages are functional. In this study, we compared the immunomodulatory effects on the CD4 T cells of M1, M2a and M2c macrophages with the macrophages that directly and indirectly cultured with MSCs. We analyzed the changes in CD14, CD64, CD80, CD163 and CD200R expression to evaluate macrophage phenotypes, and the changes in CD4, IFN-g, IL-4, IL-17a and FoxP3 expression to evaluate T helper subsets using the FACS method. The changes in IL-1b, IL-4, IL-10, IL-12p70, IL-17a and IFN-g in the media supernatants were analyzed using the Luminex method. We also performed WST-1 and Caspase-3 ELISA analyses to observe the proliferation and apoptosis status of the T cells. MSCs were found to differentiate macrophages into a distinctive phenotype, which was close to the M2c phenotype, but was not considered as an M2c cell due to the low expression of CD163, a characteristic marker for M2c. While MEM-D, MEM-ID and MSCs showed similar inhibitory effects on the Th2 and Th17 cells, the most significant increase in Treg cell frequencies was seen in MEM-D cells. Macrophages can alter their phenotypes and functions according to the stimuli from the environment. The fact that macrophages educated with MSCs suppressed the production of all the cytokines we evaluated even after the removal of MSCs suggests that these cells may be differentiated by MSCs into a suppressive macrophage subgroup. However, the Treg cell activation caused by direct interactions between MSCs and macrophage cells may be the most prominent observation of this study compared to previous work. As a result, according to our data, the interactions between MSCs and macrophages may lead to differentiation of macrophage cells into an immunosuppressive phenotype, and these macrophages may suppress the T lymphocyte subgroups at least as effectively as MSCs. However, our data obtained from in vitro experiments should be supported by future in vivo studies.

The findings of the studies on poly(ADP-ribose) polymerase-1 (PARP-1) have suggested that the enzyme inactivation provides significant protection against systemic or tissue inflammation in animal models. It has also shown that the single-nucleotide polymorphism (Val762Ala) of the PARP-1 causes about 40% decrease of enzyme activity. The aim of this study was to analyze the association of the PARP-1 Val762Ala polymorphism in Turkish patients with rheumatoid arthritis. A total of 128 RA patients and 165 normal controls from the same geographic region were studied and polymerase chain reaction (PCR)-based restriction analysis was used to identify Val762Ala polymorphism of the PARP-1. Association analyses were performed using chi (2) tests. Our results indicated that the distribution of the PARP-1 genotypes and alleles did not differ significantly among subjects with or without RA (P > 0.05). The results of the study indicate that, for our Turkish sample, the V762A polymorphism of the PARP-1 may not be involved in susceptibility to RA, implying that the polymorphism may not function as a candidate gene marker for screening RA patients.

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